Water-soluble bean-based extracts

ABSTRACT

This invention relates to a water-soluble bean-based extract including one or more isoflavone glycosides. Also included is a method of preparing such a water-soluble bean-based extract.

This application is a continuation of U.S. Ser. No. 09/464,260, filed onDec. 17, 1999.

BACKGROUND OF THE INVENTION

One of the newest and most intriguing areas of nutrition investigationinvolves food components called phytochemicals. Phytochemicals, such asisoflavones and their derivatives, are found only in plants, andalthough they are not classified as nutrients, they profoundly affectour health. See, for example, “Phytochemicals, A new paradigm” edited byW. R. Bidlack et al., published by Technomic publishing Co., Inc, 1998.

The discovery of phytochemicals has caused the dawning of a new era innutrition. The phytochemicals found in beans, such as soybeans, are ofparticular interest. Beans and the products made from them, e.g.,soybeans and soybean products, offer a unique approach to lowering theincidence of many chronic diseases such as cancer. See, for example,Journal of Agricultural Food Chemistry, 42, 1666 (1994); i.d., 43, 1184(1995). Recently, research shows that consuming just one serving of soyfoods a day may be enough to obtain the benefits of thesephytochemicals. (See “The simple soybean and your health” by M. Messinaet al., published by Avery publishing Group, Garden City Park, N.Y.,1994).

Many people, however, do not like to eat soy foods because of theirsmell, taste, and texture. One way to obviate the dislike of soy foodswould be to take soybean isoflavones as dietary supplements.Unfortunately, soybean isoflavones and their derivatives have not beenutilized as dietary supplements because of their low solubility inwater. See “Genestein” by R. Elkins, published by Woodland Publishing,Pleasant Grove, Utah 1998.

SUMMARY OF THE INVENTION

In one aspect, the invention features a water-soluble bean-based dry orwet extract including one or more isoflavone derivatives of the formula:

where R₁ is hydrogen or hydroxy, R₂ is hydrogen or methoxy, R₃ ishydrogen or CO—R₄, R₄ being methyl or carboxymethyl. The water-solublebean-based extract, in dried form, includes from about 5 to about 50parts by weight of isoflavone derivatives and has a solubility fromabout 10 mg/ml to about 1,000 mg/ml in water at about 25° C. Examples ofisoflavone derivatives present in the water-soluble bean-based extractinclude daidzin, genistin, glycitin, 6″-O-acetyldaidzin,6″-O-acetylgenistin, 6″-O-acetylglycitin, 6″-O-malonyldaidzin,6″-O-malonylgenistin, or 6″-O-malonylglycitin.

The water-soluble bean-based extract of this invention includes one ormore isoflavone derivatives, e.g., an isoflavone glycoside, whichcomplex with other components contained in beans to unexpectedly enhancetheir solubility in water.

In another aspect the invention features a method of preparing awater-soluble bean-based extract. The method includes mixing apulverized crude bean extract with water, heating the mixture, adding acoagulant to the mixture to form a suspension, heating the suspension,and collecting the supernatant to obtain a water-soluble bean-basedextract.

The water-soluble bean-based extract of this invention has a pleasanttaste and smell, and exhibits desirable composition and performance whenused as an ingredient in any food product. For example, thewater-soluble bean-based extract can be combined with a food productsuch as milk, tea, soft drink, juice, coffee, seasoning, cereal, water,beer, cookies, chewing gum, chocolate, or soup.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the HPLC chart of the major components in the water-solublebean-based extract prepared in Example 1.

DETAILED DESCRIPTION

The present invention relates to a water-soluble bean-based extractincluding isoflavone derivatives having the formula:

wherein R₁ is hydrogen or hydroxy, R₂ is hydrogen or methoxy, R₃ ishydrogen or CO—R₄, R₄ being methyl or carboxymethyl.

Examples of isoflavone derivatives present in an extract of thisinvention are daidzin, genistin, glycitin, 6″-O-acetyldaidzin,6″-O-acetylgenistin, 6″-O-acetylglycitin, 6″-O-malonyldaidzin,6″-O-malonylgenistin, and 6″-O-malonylglycitin. Isoflavone glycosides,such as daidzin and genistein, are absorbed in the stomach or smallintestine and metabolized to isoflavones via aglycones by humanintestinal microflora producing rhamnosidase, exo-beta-glucosidase,endo-beta-glucosidase or beta-glucuronidase. See, for example, Biol.Pharm. Bull, 21 (1998); Biol. Pharm. Bull, 17, 1369 (1994); and Arch.Pharm. Res., 21, 17 (1998).

The water-soluble bean-based extract of this invention has increasedwater solubility which improves the physiological absorption of theisoflavone derivatives into the body when used in food products. Forinstance, the solubility of genistin is about 5 mg/ml in methanol andabout 1 mg/ml in water at 25° C., whereas the solubility of thewater-soluble bean-based extract of this invention, containing at about10 percent of isoflavone derivatives, is about 300 mg/ml in water.Typically, the water-soluble bean-based extract of the inventionprovides an increase of about 30 fold in the solubility of isoflavonederivatives. In dried form, it includes from about 5 to about 50 partsby weight of isoflavone derivatives and has a solubility from about 10mg/ml to about 1,000 mg/ml in water at about 25° C.; preferably, about10 to about 20 parts by weight of isoflavone derivative and having asolubility from about 30 mg/ml to about 300 mg/ml; and more preferably,about 10 to about 20 parts by weight of isoflavone derivatives andhaving a solubility from about 50 mg/ml to about 200 mg/ml. Of course,the weight percentage of isoflavone derivatives affects the overallsolubility of the water-soluble bean-based extract. For instance, theoverall solubility of an extract having a higher weight percentage ofisoflavone derivatives will be less than an extract containing a lowerweight percentage of isoflavone derivatives.

Any variety of beans can be used to make the water-soluble bean-basedextract of this invention. Examples of beans used in making thewater-soluble bean-based extracts include but are not limited tosoybeans, mug beans, black beans, and kidney beans. Preferably the beansare soybeans. In general, there are two different types of soybeans,e.g., food beans and oil beans, which can be used to make thewater-soluble bean-based extract of this invention. Oil beans arepreferred since they have smaller amounts of proteins relative to foodsoybeans. Typically, oil soybeans are first processed to produce“processed” soybeans by eliminating soybean oil. Examples of processesfor eliminating soybean oil can be found in “Soybean Chemistry,Technology, and Utilization”, by K. S. Liu, Aspen Publishers, Inc.Gaithersburg, Md. 1999.

The method of preparing the water-soluble bean-based extract of thisinvention includes mixing about 1 to about 50 parts by weight of apulverized bean crude extract with about 100 parts by weight of water(hot or cold). A pulverized bean crude extract can be produced byextracting crushed beans with about 50% to about 90% aqueous alcohol or100% organic solvents, such as ethanol, methanol, or acetonitrile. Anexample of a pulverized bean crude extract is soy jam. Soy jamrepresents about 1% by weight of soybeans yet contains a higherconcentration of isoflavone derivatives relative to the remainingsoybean portions. For example, the concentration of isoflavonederivatives in soy jam is about 30 times greater than the remainingsoybean portions. See, for example, “Soybean Chemistry, Technology, andUtilization”, by K. S. Liu, Aspen Publishers, Inc. Gaithersburg, Md.1999. Pulverized bean crude extracts, such as soy jams, can be obtainedfrom Schousten USA, Inc. as SoyLife 150 and SoyLife 25. Alternatively,pulverized soybean crude extracts can be purchased from Archar DanielsMidland Company as NOVASOY.

The mixture of water and pulverized bean crude extract is then heated tobetween about 60° C. and about 100° C. for about 10 to about 120 minutesand between about 0.001 to about 2.0 parts by weight of coagulant perabout 100 parts by weight of water is added to the mixture to form asuspension, which is heated again to between about 30° C. and about 100°C. for about 1 to about 120 minutes. The supernatant is subsequentlycollected, e.g., by filtration or centrifugation, to obtain awater-soluble bean-based extract having increased solubility.Additionally, the pH of the suspension or supernatant can be adjusted byadding acids or bases to avoid precipitating isoflavone derivatives.Typically, the pH is adjusted to about 6.0

The supernatant thus obtained can be decolorized to remove anyundesirable color. Decolorizing methods include, but are not limited to,hydrogenation with the aid of a nickel catalyst, and other bleachingtechniques used in the manufacture of soybean oils. Decolorizing methodscan be found, for example, in “Soybeans, Chemistry, Technology, andUtilization” by K. Liu, published by An Aspen Publication, 1999, and “InIntroduction to oils and fats Technology” edited by P. J. Wan, p-95,published by AOCS Press, Champaign, Ill. The supernatant can also beconcentrated by any distillation system known in the art, including butnot limited to molecular distillation or evaporation. An example of amolecular distillation system is a Wiped-Film Still, available from POPESCIENTIFIC, INC, located at 351 N. Dekora Woods Blvd. Saukville, Wis.53080, USA. Additionally, the supernatant can be dried by any dryingmethod known in the art, such as lyophilization or spray-drying. (See,for example, U.S. Pat. No. 5,882,717).

The water-soluble bean-based extract of this invention can be producedby a batch method or a flow method, i.e., a continuous extraction andfiltration process. Typically, flow processes are used to help maintainreasonable manufacturing costs.

The solubility of isoflavone glycosides is enhanced by the addition ofabout 0.001 to 3.0 parts by weight of a coagulant, such as CaCl₂, CaSO₄,MgCl₂, MgSO₄, any organic or inorganic acid, (e.g., citrus juice,fumaric acid, lactic acid, malic acid, tartalic acid, H₂SO₄, H₃PO₄, orHCl), saccharin, glucono-D-lactone, and papain in 100 parts by weight ofwater. When preparing the extract, adding the coagulant tends to forminsoluble aggregates of bean proteins or polysaccharides by simplecross-linking or denaturing. The concentration of coagulant directlyaffects the amount of proteins or polysaccharides in the extract. Forexample, larger amounts of coagulant cause more insoluble aggregates toform. This effect permits control of the total amount of water-solubleproteins and polysaccharide in the extract, as well as the solubility ofthe isoflavone derivatives. The amount of coagulant to be added can bedetermined by measuring the fat, protein, and inorganic salt content ofthe resultant bean-based extract. In general, enough coagulant is addedto produce an extract having less than about 1% of fat, between about 1%to about 5% of protein, between about 0.01% to about 3% of Na, betweenabout 0.01% to about 3% of Ca, and between about 0.01% to about 3% ofany acids. The amount of fat, protein, Na, Ca, and acid in thewater-soluble bean-based extract can be determined by any knownanalytical method, such as by following the analytical methodsrecommended by the Food and Drug Administration (FDA). Typically, about0.3 parts by weight of citric acid is added to the extract. In general,if too much coagulant is added, the water-soluble bean proteins,polysaccharides, or isoflavone derivatives co-precipitate out of theextract thereby decreasing the concentration of isoflavone derivativesin the extract or the solubility of the isoflavone derivatives. If toolittle coagulant is added, the bean-based mixture forms a colloid fromwhich the water-soluble bean-based extract is not easily separated.

The water-soluble bean-based extract of this invention can be added to afood product either in dried or wet form. The food product can be asolid, a paste, or a liquid food product, such as milk, tea, softdrinks, juices, coffee, seasonings, cereals, water, beer, cookies,chewing gum, chocolate, or soups.

The water-soluble bean-based extract of this invention can also includeco-extracts from other grains, such as barley, rice, and malts. Forexample, one or more other grains can be processed together with beansby the methodology described above to produce a water-soluble bean-basedextract of this invention. Additionally the water-soluble bean-basedextract can be fortified with electrolytes, flavors, preservatives, andother additives, (e.g., vitamin supplements and maltodextrin). Examplesof preservatives include, but are not limited to, ascorbic acid andpropyl gallate. Examples of electrolytes include, but are not limitedto, magnesium sulfate and potassium chloride.

Without further elaboration, it is believed that the above descriptionhas adequately enabled the present invention. The following specificexamples are, therefore, to be construed as merely illustrative, and notlimitative of the remainder of the disclosure in any way whatsoever. Allof the publications cited herein, including patents, are herebyincorporated by reference in their entirety.

EXAMPLE 1

Pulverized soybean crude product (1 kg) extracted with about 90% aqueousEtOH, i.e., SoyLife 150 purchased from Schousten USA, Inc., was added to3 liters of hot water at 70° C. The mixture was stirred at 90° C. for 30min and then at 50° C. for 30 min. The residue and solution were roughlyseparated by centrifuging the mixture at 10,000×g at 50° C. Afterdecanting, 6 g of CaCl₂, food additive grade (about 0.02 M), was addedto the supernatant and additional stirring was continued at 30° C. for30 min. The supernatant was filtered through a 1-μm pore sizemicrofilter and yielded a yellow solution (2.5 liters). The filtrate wasthen transferred into a 5-liter glass flask and was hydrogenated in thepresence of catalytic amounts (about 3 grams) of 5% Pd/C at 10 atm ofhydrogen for 5 hrs. The decolorized mixture was microfiltered (0.2-μmpore size) to yield a clear water-soluble soybean-based extract solution(2.3 liters).

The water-soluble soybean-based extract solution was characterized byhigh-pressure liquid chromatography (HPLC). The HPLC was conducted on aC18-reverse phase column 150 mm ×4.6 mm, Symmetry Shield RP18 availablefrom Waters, using a gradient elution method, lml/min for 20 minuteswith 15%-40% aqueous acetonitrile and 0.1% trifluoroacetic acid at 30°C. The HPLC detector was set at 254 nm. FIG. 1 shows the HPLC chart ofthe major components in the soluble soybean product in Example 1. eachof the peaks from the HPLC chart is listed in table 1 below along withtheir retention time, peak area, and percent area. Several individualcomponents of the extract were identified by comparing the retentiontimes of the unknown components to retention times of known isoflavoneglycosides analyzed by HPLC under the same experimental conditions.

The water-soluble soybean-based extract solution was also characterizedto determine the relative amounts of the components of the extract. Asample of the extract was sent to and analyzed by Food ProductsLaboratory Inc., located in Portland, Oreg., USA, using FDA approvedmethods. The extract included <0.1% fat,5% protein,65% carbohydratesides), 20% isoflavones, 8% minerals, and 1% moisture.

TABLE 1 HPLC analysis of soluble soybean product in Example 1 PeakRetention Time Peak Area Area # (min) (mAU * sec) (%) Compound 1 6.94056.95 0.35 N.D. 2 7.231 85.42 0.53 N.D. 3 7.661 164.09 1.02 N.D. 4 8.0905129.91 31.85 Daidzin 5 8.507 2722.58 16.90 Glycitin 6 10.376 19.68 0.12N.D. 7 10.652 23.89 0.15 N.D. 8 11.069 34.82 0.22 N.D. 9 11.685 367.982.28 N.D. 10 11.990 1378.49 8.56 Genistin 11 12.766 75.19 0.47 MalonylDaidzin 12 13.048 52.95 0.33 Maionyl Glycitin 13 13.461 3038.54 18.86Acetyl Daidzin 14 13.771 1421.88 8.83 Acetyl Genistin 15 15.723 33.080.21 N.D. 16 15.942 43.63 0.27 N.D. 17 17.265 58.45 0.36 N.D. 18 17.737867.85 5.39 Acetyl Glycitin 19 18.236 288.12 1.79 Glycitein 20 18.822196.99 1.22 Daidzein 21 25.657 47.89 0.30 Genistein Total 16108.41100.00 N.D.: Not Determined

EXAMPLE 2

Pulverized soybean crude product (1 kg) extracted with about 90% aqueousEtOH, i.e., SoyLife 150 purchased from Schousten USA, Inc., was added to27 liters of hot water. The mixture was stirred at 90° C. for 15 min andthen an aqueous solution (3 liters) of citric acid (90 g) was added tothe mixture. The mixture was stirred at 90° C. for 15 min. The residueand solution were filtered through a Celite bed on paper filter undervacuum. The filtrate was a pale yellow clear solution (27 liters).Potassium carbonate (50 g) was added to the filtrate to adjust pH at6.0. The filtrate was purified to remove bacteria by further filteringthe filtrate with a 0.45 μM pore size filter (Millipore Corporation)under vacuum. The purified filtrate was then concentrated by using 2inches wiped-film stills (Pope Scientific Inc.) at 70° C. under 20 mmtorr to yield a light yellow water-soluble soybean based extractsolution (18 liters).

EXAMPLE 3

A clear water-soluble soybean-based extract (2 liters) obtained in thesame manner as described in Example 1. The extract solution was thenlyophilized using a lyophilizer manufactured by Bertis Corp for 2 daysat −85° C. under 10 mm Torr, resulting in 350 g of a pale yellow drycake. The cake, which contained 70 g of isoflavone derivatives, had asolubility of approximately 200 mg/ml. The solubility was tested byfirst adding 1 g of the dried extract into 1 ml of pure water at 25° C.with agitation for 5 min. If the extract was not completely dissolved(visual inspection), another 1 ml of water was added and the resultingmixture agitated for 5 minutes at 25° C. Next, the mixture was visuallyinspected to determine if the extract had completely dissolved. If not,water at 25° C. was added in 1 ml increments and agitated for 5 minutesafter each addition of water until the extract was completely dissolvedby visual inspection.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

Furthermore, from the above description, one skilled in the art caneasily ascertain the essential characteristics of the present invention,and without departing from the spirit and scope thereof, can makevarious changes and modifications of the invention to adapt it tovarious usages and conditions. Thus, other embodiments are also withinthe claims.

What is claimed is:
 1. A water-soluble bean-based extract comprising oneor more isoflavone derivatives of the formula:

wherein R₁ is hydrogen or hydroxy, R₂ is hydrogen or methoxy, R₃ ishydrogen or CO—R₄, R₄ being methyl or carboxymethyl; and thewater-soluble bean-based extract, in dried form, includes from about 5to about 50 parts by weight of isoflavone derivatives and has asolubility from about 10 mg/ml to about 1,000 mg/ml in water at about25° C.
 2. The water-soluble bean-based extract of claim 1, wherein thebean-based extract is based on soybeans, mug beans, black beans, orkidney beans.
 3. The water-soluble bean-based extract of claim 1,wherein the water-soluble bean-based extract, in dried form, includesfrom about 10 to 20 parts by weight of isoflavone derivatives and has asolubility from about 30 mg/ml to about 300 mg/ml in water at about 25°C.
 4. The water-soluble bean-based extract of claim 1, wherein thewater-soluble bean-based extract, in dried form, includes from about 10to about 20 parts by weight of isoflavone derivatives and has asolubility from about 50 mg/ml to about 200 mg/ml in water at about 25°C.
 5. The water-soluble bean-based extract of claim 1, wherein theisoflavone derivative is daidzin, genistin, glycitin,6″-O-acetyldaidzin, 6″-O-acetylgenistin, 6″-O-acetylglycitin,6″-O-malonyldaidzin, 6″-O-malonylgenistin, or 6″-O-malonylglycitin. 6.The water-soluble bean-based extract of claim 5, wherein thewater-soluble bean-based extract, in dried form, includes from about 10to about 20 parts by weight of isoflavone derivatives and has asolubility from about 30 mg/ml to about 300 mg/ml in water at about 25°C.
 7. The water-soluble bean-based extract of claim 5, wherein thewater-soluble bean-based extract, in dried form, includes from about 10to about 20 parts by weight of isoflavone derivatives and has asolubility from about 50 mg/ml to about 200 mg/ml in water at about 25°C.
 8. A water-soluble bean-based extract prepared by a methodcomprising: mixing a pulverized crude bean-based extract with water;heating the mixture; adding a coagulant to the mixture to form asuspension; heating the suspension; and collecting the supernatant toobtain a water-soluble bean-based extract; the water-soluble bean-basedextract containing one or more isoflavone derivatives the formula:

wherein R₁ is hydrogen or hydroxy, R₂ is hydrogen or methoxy, R₃ ishydrogen or CO—R₄, R₄ being methyl or carboxymethyl; and thewater-soluble bean-based extract, in dried form, includes from about 5to about 50 parts by weight of isoflavone derivatives and has asolubility from about 10 mg/ml to about 1,000 mg/ml in water at about25° C.
 9. The water-soluble bean-based extract of claim 8, wherein thebean-based extract is based on soybeans, mug beans, black beans, orkidney beans.
 10. The water-soluble bean-based extract of claim 8,wherein about 0.01 to about 3 parts by weight of the coagulant is addedto about 100 parts by weight of water.
 11. The water-soluble bean-basedextract of claim 10, wherein the coagulant is citric acid.
 12. Thewater-soluble bean-based extract of claim 8, wherein the supernatant iscollected by filtering the suspension.
 13. The water-soluble bean-basedextract of claim 8, wherein the method further comprising drying thesupernatant to obtain a dried water-soluble bean-based extract.
 14. Awater-soluble bean-based extract prepared by a method comprising: mixingabout 1 to about 50 parts by weight of a pulverized crude bean-basedextract with about 100 parts by weight of water; heating the mixture tobetween about 60° C. and about 100° C. for about 10 to about 120minutes; adding a coagulant to the mixture to form a suspension; heatingthe suspension to between about 30° C. and about 100° C. for about 1 toabout 120 minutes; and collecting the supernatant to obtain awater-soluble bean-based extract.
 15. The water-soluble bean-basedextract of claim 14, wherein the bean-based extract is based onsoybeans, mug beans, black beans, or kidney beans.
 16. A food productcomprising the water-soluble bean-based extract of claim
 1. 17. The foodproduct of claim 16, wherein the bean-based extract is based onsoybeans, mug beans, black beans, or kidney beans.
 18. A food productcomprising the water-soluble bean-based extract of claim
 7. 19. A foodproduct comprising the water-soluble bean-based extract of claim
 8. 20.The food product of claim 16, wherein the food product is milk, tea,soft drink, juice, coffee, seasoning, cereal, water, beer, cookies,chewing gum, chocolate, or soup.
 21. The food product of claim 17,wherein the food product is milk, tea, soft drink, juice, coffee,seasoning, cereal, water, beer, cookies, chewing gum, chocolate, orsoup.
 22. The food product of claim 18, wherein the food product ismilk, tea, soft drink, juice, coffee, seasoning, cereal, water, beer,cookies, chewing gum, chocolate, or soup.
 23. The food product of claim19, wherein the food product is milk, tea, soft drink, juice, coffee,seasoning, cereal, water, beer, cookies, chewing gum, chocolate, orsoup.
 24. The water-soluble bean-based extract of claim 8, wherein thebean-based extract is based on soybeans, mug beans, black beans, orkidney beans.
 25. The water-soluble bean-based extract of claim 8,wherein the water-soluble bean-based extract, in dried form, includesfrom about 10 to 20 parts by weight of isoflavone derivatives and has asolubility from about 30 mg/ml to about 300 mg/ml in water at about 25°C.
 26. The water-soluble bean-based extract of claim 8, wherein thewater-soluble bean-based extract, in dried form, includes from about 10to about 20 parts by weight of isoflavone derivatives and has asolubility from about 50 mg/ml to about 200 mg/ml in water at about 25°C.
 27. The water-soluble bean-based extract of claim 8, wherein theisoflavone derivative is daidzin, genistin, glycitin,6″-O-acetyldaidzin, 6″-O-acetylgenistin, 6″-O-acetylglycitin,6″-O-malonyldaidzin, 6″-O-malonylgenistin, or 6″-O-malonylglycitin.